Phosphorylation of recombinant mouse cell division cycle 25 homolog(CDC25B) protein in vitro

Objective To verify mouse CDC25B acting as a direct protein kinase A(PKA) substrate by phosphorylation in vitro and autoradiography.Methods Prokaryotic expression of the pGEX-4T-2-CDC25B₂₀₁ fusion protein was expressed in the presence of isopropyl-β-D thiogalactopyranoside(IPTG) and purified by the glutathione sepharose 4B protein with chromatography and phosphorylated with PKA in vitro,and then identified by sodium dodecyl sulfate polyarylamide gel electropheresis (SDS-PAGE),western blotting,and autoradiography.Results The glutathione S-transferase (GST) CDC25B₂₀₁ was expressed highly for 3 hours at 27℃ in Escherichia coli BL21 (DE3) transformed with pGEX-4T-2CDC25B 201 in the presence of 0.1mmol/L IPTG.The results of SDS-PAGE show ed a clear band of GST-CDC25B 201 fusion protein in 50 kD of the expected size,whereas the band of phosphorylated GST-CDC25B₂₀₁ protein was seen at about 55kD by autoradiography.Total protein induced by IPTG,vector-expressed protein,and purified protein were identified by western blotting and GST bands were emerged in corresponding position.Conclusion Mouse CDC25B is the direct dow nstream substrate of PKA.